EP2365822A1 - Facteur v de serpent et procédés d'utilisation en tant que procoagulant - Google Patents

Facteur v de serpent et procédés d'utilisation en tant que procoagulant

Info

Publication number
EP2365822A1
EP2365822A1 EP09828344A EP09828344A EP2365822A1 EP 2365822 A1 EP2365822 A1 EP 2365822A1 EP 09828344 A EP09828344 A EP 09828344A EP 09828344 A EP09828344 A EP 09828344A EP 2365822 A1 EP2365822 A1 EP 2365822A1
Authority
EP
European Patent Office
Prior art keywords
nucleic acid
derivative
rfv
factor
protein
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
EP09828344A
Other languages
German (de)
English (en)
Other versions
EP2365822B1 (fr
EP2365822A4 (fr
Inventor
Rodney M. Camire
Mettine H. A. BOS
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Childrens Hospital of Philadelphia CHOP
Original Assignee
Childrens Hospital of Philadelphia CHOP
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Childrens Hospital of Philadelphia CHOP filed Critical Childrens Hospital of Philadelphia CHOP
Publication of EP2365822A1 publication Critical patent/EP2365822A1/fr
Publication of EP2365822A4 publication Critical patent/EP2365822A4/fr
Application granted granted Critical
Publication of EP2365822B1 publication Critical patent/EP2365822B1/fr
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/745—Blood coagulation or fibrinolysis factors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates the fields of medicine and hematology. More specifically, the invention describes therapeutic strategies using snake venom derived variants of Factor V (FV) and derivatives thereof for modulating the coagulation cascade in patients in need thereof.
  • FV Factor V
  • Blood coagulation FV is a multi-domain protein (A1-A2-B-A3-C1-C2) that circulates as a procofactor with little or no procoagulant activity. Conversion to the active cofactor state (FVa) involves proteolytic removal of a large, heavily glycosylated central B-domain mediated by thrombin (1). FVa reversibly associates with the serine protease factor Xa (FXa) on a negatively charged membrane surface in the presence of calcium ions to form prothrombinase, the physiological activator of prothrombin (2).
  • FXa serine protease factor Xa
  • Venom-derived FV from each of these species shares ⁇ 44% sequence homology with mammalian FV having a similar domain structure (7-9). However, the B-domain length is dramatically shortened compared to human FV (-46 versus 836 residues).
  • the venom from these snakes is considered the most toxic in the world, is strongly procoagulant and induces, among other symptoms, disseminated intravascular coagulopathy (10). It contains large amounts of FV as well as a FXa-like enzyme, together forming a complex which is a powerful prothrombin activator. This complex from O.
  • scutellatus oscutarin C
  • P. textilis pseutarin C
  • APC activated protein C
  • a method for the treatment of a blood coagulation disorder in a patient in need thereof entails administering an effective amount of an activated form of FV variant or a derivative thereof, thereby enhancing clot formation in said patient and ameliorating the symptoms of the bleeding disorder.
  • the variant factor V is encoded by a nucleic acid having the sequence of SEQ ID NO: 1.
  • the variant Factor V is encoded by the codon optimized nucleic acid having the sequence of SEQ ID NO: 2.
  • the method and Factor V pro-coagulant can be used for the treatment of blood coagulation disorders which include, without limitation, hemophilia A and B, hemophilia A and B associated with inhibitory antibodies, coagulation factor deficiency, vitamin K epoxide reductase Cl deficiency, gamma-carboxylase deficiency, bleeding associated with trauma, injury, thrombosis, thrombocytopenia, stroke, coagulopathy, disseminated intravascular coagulation (DIC); over- anticoagulation treatment disorders, Bernard Soulier syndrome, Glanzman thromblastemia, and storage pool deficiency.
  • blood coagulation disorders which include, without limitation, hemophilia A and B, hemophilia A and B associated with inhibitory antibodies, coagulation factor deficiency, vitamin K epoxide reductase Cl deficiency, gamma-carboxylase deficiency, bleeding associated with trauma, injury, thrombosis, thrombocytopenia
  • the blood coagulation disorder may be due to administration of heparin, low molecular weight heparin, pentasaccharide, warfarin, small molecule antithrombotics and FXa inhibitors.
  • the variant Factor V of the invention may be directly infused or administered topically to a patient or may be delivered in the form of a vector encoding the same.
  • a humanized and improved Factor V variant which exhibits one or more of the following properties: the FV variant is synthesized in an active, cofactor state; the FV variant possesses a unique conformation such as to allow it to bypass the normal requirement for a membrane surface to achieve high affinity binding to its serine protease factor Xa (FXa); and the FV variant is functionally resistant to inactivation by activated protein C (APC), via inclusion of a unique disulfide bond connecting its heavy and light chains.
  • APC activated protein C
  • an isolated nucleic acid of SEQ ID NO: 2 encoding snake Factor V is provided.
  • Such nucleic acids can be contained in an expression vector for use.
  • the invention provides an isolated snake Factor V protein encoded by the nucleic acid of SEQ ID NO: 1.
  • the snake Factor V protein can be contained in a liposome or micelle.
  • the invention provides a pharmaceutical composition comprising the snake Factor V protein contained in a pharmaceutically acceptable excipient.
  • FIG. 1 SDS-PAGE analysis of purified proteins. Proteins (3 ⁇ g/lane) were subjected to SDS-PAGE under reducing (panels A, C) or non-reducing (panel B) conditions and visualized by staining with Coomassie Brilliant Blue R-250. Lane 1, pt-rFV; lane 2, pt-rFV-QQ; lane 3, pt-rFV plus thrombin; lane 4, pt-rFV-QQ plus thrombin, lane 5, rFX; lane 6, rFXa; lane 7, pt-FXa. The apparent molecular weights of the standards are indicated on the left. Panels A and B share the same markers. N-terminal sequence results of the indicated protein bands are shown. The dash (-) as position six indicates that the yield was too low to accurately assign an amino acid and likely represents the presence of ⁇ -carboxyglutamic acid.
  • Figure 2 Determination of kinetic constants for cleavage of prothrombin in the presence or absence of PCPS.
  • the initial velocity of thrombin generation was determined at increasing concentrations of prothrombin in the presence (panel A) or absence (panel B) of 50 ⁇ M PCPS and 3 ⁇ M DAPA with 0.1 nM pt-FXa and 20 nM hFV-810 (- ⁇ - or -O-), pt-rFV (-•- or -O-), pt-rFV-QQ (-A- or - ⁇ -), or pt-rFVa (- ⁇ - or -D-).
  • the initial velocity of thrombin generation was determined at increasing concentrations of prethrombin-1 in the presence (panel A) or absence (panel B) of 50 ⁇ M PCPS and 3 ⁇ M DAPA with 0.1 nM pt-FXa and 20 nM pt-rFV (-•- or -O-), pt-rFV-QQ (-A- or - ⁇ -), or pt-rFVa (- ⁇ - or -D-).
  • the lines were drawn following analysis of all data sets to a rectangular hyperbola, and the fitted kinetic constants can be found in Table 2. The data are representative of two similar experiments.
  • FIG. 4 Inactivation of membrane-bound FV(a) by APC. Reaction mixtures containing 50 ⁇ M PCPS and 500 nM pt-rFV (-•-), pt-rFVa (-D-), hFV-810 (- ⁇ -), or rFVa (- ⁇ -) were incubated with either 10 nM or 750 nM (inset) APC. At selected time intervals, samples were removed and assayed for cofactor activity as described in "Experimental Procedures" (panel A) or run on a SDS-PAGE gel and stained with Coomassie Blue R250 under reducing (panel B) or non- reducing conditions (panel C). The data are representative of two similar experiments.
  • FIG. 5 Nucleic Acid Coding sequence for Factor V.
  • the nucleic acid sequence of the snake venom derived Factor V (SEQ ID NO: 1) is shown in panels 5 A and 5B.
  • the nucleic acid sequence of the snake venom derived Factor V (SEQ ID NO: 2) which has been codon optimized for expression in mammalian systems is shown in panels 5C and 5D.
  • Venomous snakes produce a diverse array of toxic compounds, including procoagulants to defend themselves and incapacitate prey.
  • the Australian brown snake Pseudonaja textilis has a venom-derived prothrombin activator complex which is homologous to factor Xa (FXa) and factor V (FV).
  • the FV component (pt-FV) has a similar domain organization as human FV however its central B-domain is much smaller (55 versus 836 amino acids).
  • pt-FV has acquired unique gain of function elements transforming it into a potent procoagulant.
  • Measurements using recombinant pt-FV revealed that it is synthesized as a constitutively active cofactor, which unlike human FV, does not require proteolysis to express activity.
  • the invention also provides a unique derivative of factor V (FV) which is expressed in the venom of the common brown snake P. textilis or heterologous expression systems (pt-FV) or a human FV derivative expressed in similar systems that has one or more of the following unique characteristics: a) a FV variant that is synthesized in an active, cofactor state, b) a FV variant that has a unique conformation such as to allow it to bypass the normal requirement for a membrane surface to achieve high affinity binding to its serine protease factor Xa (FXa); c) a FV variant that is rendered functionally resistant to inactivation by activated protein C (APC), via inclusion of a unique disulfide bond connecting its heavy and light chains.
  • FV factor V
  • APC activated protein C
  • variant FV refers to a derivative FV molecule obtainable from snake or other heterologous expression systems which exhibits potent procoagulant activity.
  • the variant FV of the invention encoded by SEQ ID NO: 1 or variations thereof (i.e. SEQ ID NO: 2) are compatible for use in humans and promotes desirable FV procoagulant activity.
  • hemophilia A and B hemophilia A and B patients with inhibitory antibodies, deficiencies in coagulation Factors, VIIl IX and X, XI, V, XII, II, von Willebrand factor, combined
  • FV/FV1 deficiency vitamin K epoxide reductase Cl deficiency, gamma-carboxylase deficiency; bleeding associated with trauma, injury, thrombosis, thrombocytopenia, stroke, coagulopathy, disseminated intravascular coagulation (DIC); over-anticoagulation associated with heparin, low molecular weight heparin, pentasaccharide, warfarin, small molecule antithrombotics (i.e. FXa inhibitors); and platelet disorders such as, Bernard Soulier syndrome, Glanzman thromblastemia, and storage pool deficiency.
  • isolated nucleic acid refers to a DNA molecule that is separated from sequences with which it is immediately contiguous (in the 5' and 3' directions) in the naturally occurring genome of the organism from which it originates.
  • the "isolated nucleic acid” may comprise a DNA or cDNA molecule inserted into a vector, such as a plasmid or virus vector, or integrated into the DNA of a prokaryote or eukaryote.
  • RNA molecules of the invention the term “isolated nucleic acid” primarily refers to an RNA molecule encoded by an isolated DNA molecule as defined above.
  • the term may refer to an RNA molecule that has been sufficiently separated from RNA molecules with which it would be associated in its natural state (i.e., in cells or tissues), such that it exists in a “substantially pure” form (the term “substantially pure” is defined below).
  • the term “isolated protein” or “isolated and purified protein” is sometimes used herein. This term refers primarily to a protein produced by expression of an isolated nucleic acid molecule of the invention. Alternatively, this term may refer to a protein which has been sufficiently separated from other proteins with which it would naturally be associated, so as to exist in "substantially pure” form.
  • promoter region refers to the transcriptional regulatory regions of a gene, which may be found at the 5 ' or 3' side of the coding region, or within the coding region, or within introns.
  • vector refers to a small carrier DNA molecule into which a DNA sequence can be inserted for introduction into a host cell where it will be replicated.
  • An "expression vector” is a specialized vector that contains a gene or nucleic acid sequence with the necessary regulatory regions needed for expression in a host cell.
  • operably linked means that the regulatory sequences necessary for expression of a coding sequence are placed in the DNA molecule in the appropriate positions relative to the coding sequence so as to effect expression of the coding sequence.
  • This same definition is sometimes applied to the arrangement of coding sequences and transcription control elements (e.g. promoters, enhancers, and termination elements) in an expression vector. This definition is also sometimes applied to the arrangement of nucleic acid sequences of a first and a second nucleic acid molecule wherein a hybrid nucleic acid molecule is generated.
  • transcription control elements e.g. promoters, enhancers, and termination elements
  • substantially pure refers to a preparation comprising at least 50- 60% by weight the compound of interest (e.g., nucleic acid, oligonucleotide, protein, etc.). More preferably, the preparation comprises at least 75% by weight, and most preferably 90-99% by weight, of the compound of interest. Purity is measured by methods appropriate for the compound of interest (e.g. chromatographic methods, agarose or polyacrylamide gel electrophoresis, HPLC analysis, and the like).
  • phrases "consisting essentially of when referring to a particular nucleotide sequence or amino acid sequence means a sequence having the properties of a given SEQ ID NO:.
  • the phrase when used in reference to an amino acid sequence, the phrase includes the sequence per se and molecular modifications that would not affect the basic and novel characteristics of the sequence.
  • oligonucleotide refers to primers and probes of the present invention, and is defined as a nucleic acid molecule comprised of two or more ribo- or deoxyribonucleotides, preferably more than three. The exact size of the oligonucleotide will depend on various factors and on the particular application for which the oligonucleotide is used.
  • probe refers to an oligonucleotide, polynucleotide or nucleic acid, either RNA or DNA, whether occurring naturally as in a purified restriction enzyme digest or produced synthetically, which is capable of annealing with or specifically hybridizing to a nucleic acid with sequences complementary to the probe.
  • a probe may be either single-stranded or double- stranded. The exact length of the probe will depend upon many factors, including temperature, source of probe and method of use. For example, for diagnostic applications, depending on the complexity of the target sequence, the oligonucleotide probe typically contains 15-25 or more nucleotides, although it may contain fewer nucleotides.
  • the probes herein are selected to be “substantially” complementary to different strands of a particular target nucleic acid sequence. This means that the probes must be sufficiently complementary so as to be able to "specifically hybridize” or anneal with their respective target strands under a set of pre-determined conditions. Therefore, the probe sequence need not reflect the exact complementary sequence of the target. For example, a non-complementary nucleotide fragment may be attached to the 5 ' or 3' end of the probe, with the remainder of the probe sequence being complementary to the target strand. Alternatively, non-complementary bases or longer sequences can be interspersed into the probe, provided that the probe sequence has sufficient complementarity with the sequence of the target nucleic acid to anneal therewith specifically.
  • the term “specifically hybridize” refers to the association between two single- stranded nucleic acid molecules of sufficiently complementary sequence to permit such hybridization under predetermined conditions generally used in the art (sometimes termed “substantially complementary”).
  • the term refers to hybridization of an oligonucleotide with a substantially complementary sequence contained within a single-stranded DNA or RNA molecule of the invention, to the substantial exclusion of hybridization of the oligonucleotide with single-stranded nucleic acids of non-complementary sequence.
  • primer refers to an oligonucleotide, either RNA or DNA, either single-stranded or double-stranded, either derived from a biological system, generated by restriction enzyme digestion, or produced synthetically which, when placed in the proper environment, is able to act functionally as an initiator of template-dependent nucleic acid synthesis.
  • suitable nucleoside triphosphate precursors of nucleic acids, a polymerase enzyme, suitable cofactors and conditions such as a suitable temperature and pH
  • the primer may be extended at its 31 terminus by the addition of nucleotides by the action of a polymerase or similar activity to yield a primer extension product.
  • the primer may vary in length depending on the particular conditions and requirements of the application.
  • the oligonucleotide primer is typically 15-25 or more nucleotides in length.
  • the primer must be of sufficient complementarity to the desired template to prime the synthesis of the desired extension product, that is, to be able to anneal with the desired template strand in a manner sufficient to provide the 3' hydroxyl moiety of the primer in appropriate juxtaposition for use in the initiation of synthesis by a polymerase or similar enzyme. It is not required that the primer sequence represent an exact complement of the desired template.
  • a non-complementary nucleotide sequence may be attached to the 5' end of an otherwise complementary primer.
  • non-complementary bases may be interspersed within the oligonucleotide primer sequence, provided that the primer sequence has sufficient complementarity with the sequence of the desired template strand to functionally provide a template-primer complex for the synthesis of the extension product.
  • the Blastn 2.0 program provided by the National Center for Biotechnology Information (on the world wide web at .ncbi.nlm.nih.gov/blast/; Altschul et al., 1990, J MoI Biol 215:403-410) using a gapped alignment with default parameters, may be used to determine the level of identity and similarity between nucleic acid sequences and amino acid sequences.
  • Nucleic acid molecules encoding the variant forms of FV or functional derivatives thereof of the invention may be prepared by using recombinant DNA technology methods. The availability of nucleotide sequence information enables preparation of isolated nucleic acid molecules of the invention by a variety of means. For example, nucleic acid sequences encoding the variant forms of FV or derivative thereof polypeptide may be isolated from appropriate biological sources using standard protocols well known in the art. Nucleic acids of the present invention may be maintained as DNA in any convenient cloning vector. In a preferred embodiment, clones are maintained in a plasmid cloning/expression vector, such as pBluescript (Stratagene, La Jolla, CA), which is propagated in a suitable E. coli host cell.
  • pBluescript Stratagene, La Jolla, CA
  • the nucleic acids may be maintained in vector suitable for expression in mammalian cells.
  • the variant FV or derivative thereof-encoding nucleic acid molecules of the invention include cDNA, genomic DNA, RNA, and fragments thereof which may be single- or double- stranded.
  • this invention provides oligonucleotides (sense or antisense strands of DNA or RNA) having sequences capable of hybridizing with at least one sequence of a nucleic acid molecule of the present invention. Such oligonucleotides are useful as probes for detecting the FV expression.
  • a full-length variant of FV or derivative thereof polypeptide of the present invention may be prepared in a variety of ways, according to known methods.
  • the protein may be purified from appropriate sources, e.g., transformed bacterial or animal cultured cells or tissues which express the activated form of FV, by immunoaffinity purification. However, this is not a preferred method due to the low amount of protein likely to be present in a given cell type at any time.
  • the availability of nucleic acid molecules encoding a variant form of FV or derivative thereof polypeptide enables production of the variant form of FV or derivative thereof using in vitro expression methods known in the art.
  • a cDNA or gene may be cloned into an appropriate in vitro transcription vector, such as pSP64 or pSP65 for in vitro transcription, followed by cell-free translation in a suitable cell-free translation system, such as wheat germ or rabbit reticulocyte lysates.
  • in vitro transcription and translation systems are commercially available, e.g., from Promega Biotech, Madison, Wisconsin or BRL, Rockville, Maryland.
  • FV or derivative thereof may be produced by expression in a suitable prokaryotic or eukaryotic expression system.
  • a DNA molecule encoding the variant form of FV for example, may be inserted into a plasmid vector adapted for expression in a bacterial cell, such as E. coli or a mammalian cell such as CHO or HeIa cells.
  • tagged fusion proteins comprising the variant form of FV or derivative thereof can be generated.
  • Such FV molecules or derivative thereof-tagged fusion proteins are encoded by part or all of a DNA molecule, ligated in the correct codon reading frame to a nucleotide sequence encoding a portion or all of a desired polypeptide tag which is inserted into a plasmid vector adapted for expression in a bacterial cell, such as E. coli or a eukaryotic cell, such as, but not limited to, yeast and mammalian cells.
  • Vectors such as those described above comprise the regulatory elements necessary for expression of the DNA in the host cell positioned in such a manner as to permit expression of the DNA in the host cell.
  • regulatory elements required for expression include, but are not limited to, promoter sequences, transcription initiation sequences, and enhancer sequences.
  • the snake venom derived variant FV or derivative thereof protein, produced by gene expression in a recombinant prokaryotic or eukaryotic system may be purified according to methods known in the art.
  • a commercially available expression/secretion system can be used, whereby the recombinant protein is expressed and thereafter secreted from the host cell, to be easily purified from the surrounding medium.
  • an alternative approach involves purifying the recombinant protein by affinity separation, such as by immunological interaction with antibodies that bind specifically to the recombinant protein or nickel columns for isolation of recombinant proteins tagged with 6-8 histidine residues at their N-terminus or C-terminus.
  • Alternative tags may comprise the FLAG epitope, GST or the hemagglutinin epitope. Such methods are commonly used by skilled practitioners.
  • the FV or derivative thereof proteins, prepared by the aforementioned methods, may be analyzed according to standard procedures. For example, such proteins may be subjected to amino acid sequence analysis, according to known methods.
  • a convenient way of producing a polypeptide according to the present invention is to express nucleic acid encoding it, by use of the nucleic acid in an expression system.
  • a variety of expression systems of utility for the methods of the present invention are well known to those of skill in the art.
  • the present invention also encompasses a method of making a polypeptide (as disclosed), the method including expression from nucleic acid encoding the polypeptide (generally nucleic acid). This may conveniently be achieved by culturing a host cell, containing such a vector, under appropriate conditions which cause or allow production of the polypeptide. Polypeptides may also be produced in in vitro systems, such as reticulocyte lysate. III. Uses of FV Encoding Nucleic Acids and Proteins or Derivatives Thereof
  • the FV polypeptide or derivative thereof or nucleic acids encoding the same having altered coagulation activities may be used according to this invention, for example, as therapeutic and/or prophylactic agents which modulate the blood coagulation cascade.
  • the present inventors have discovered that these molecules can be altered to increase coagulation.
  • the FV or derivative thereof may be administered to a patient via infusion in a biologically compatible carrier, preferably via intravenous injection.
  • the FV or derivative thereof of the invention may optionally be encapsulated into liposomes or mixed with other phospholipids or micelles to increase stability of the molecule.
  • the FV or derivative thereof may be administered alone or in combination with other agents known to modulate hemostasis (e.g., Factor Vila, FIX, FVIII or FX/Xa and derivatives thereof).
  • An appropriate composition in which to deliver FV or derivative thereof may be determined by a medical practitioner upon consideration of a variety of physiological variables, including, but not limited to, the patient's condition and hemodynamic state.
  • the preparation containing the purified FV or derivative thereof contains a physiologically acceptable matrix and is preferably formulated as a pharmaceutical preparation.
  • the preparation can be formulated using substantially known prior art methods, it can be mixed with a buffer containing salts, such as NaCl, CaCl 2 , and amino acids, such as glycine and/or lysine, and in a pH range from 6 to 8.
  • the purified preparation containing the factor V or derivative thereof can be stored in the form of a finished solution or in lyophilized or deep-frozen form.
  • the preparation is stored in lyophilized form and is dissolved into a visually clear solution using an appropriate reconstitution solution,
  • the preparation according to the present invention can also be made available as a liquid preparation or as a liquid that is deep-frozen.
  • the preparation according to the present invention is especially stable, i.e., it can be allowed to stand in dissolved form for a prolonged time prior to application.
  • the preparation according to the present invention can be made available as a pharmaceutical preparation with the FV or derivative thereof in the form of a one-component preparation or in combination with other factors in the form of a multi -component preparation.
  • the purified protein Prior to processing the purified protein into a pharmaceutical preparation, the purified protein is subjected to the conventional quality controls and fashioned into a therapeutic form of presentation. In particular, during the recombinant manufacture, the purified preparation is tested for the absence of cellular nucleic acids as well as nucleic acids that are derived from the expression vector, preferably using a method, such as is described in EP 0 714 987.
  • Another feature of this invention relates to making available a preparation which contains FV or derivative thereof with a high stability and structural integrity and which, in particular, is free from inactive factor VAVa analog intermediates and autoproteolytic degradation products and which can be produced by activating a factor V analog of the type described above and by formulating it into an appropriate preparation.
  • the pharmaceutical preparation may contain dosages of between 10- 1000 ⁇ g/kg, more preferably between about 10-250 ⁇ g/kg and most preferably between 10 and 75 ⁇ g/kg, with 40 ⁇ g/kg of the variant factor V polypeptide being particularly preferred.
  • Patients may be treated immediately upon presentation at the clinic with a bleed. Alternatively, patients may receive a bolus infusion every one to three hours, or if sufficient improvement is observed, a once daily infusion of FV or derivative thereof described herein.
  • the FV or derivative thereof-encoding nucleic acids obtained from snake may be used for a variety of purposes in accordance with the present invention.
  • a nucleic acid delivery vehicle i.e., an expression vector
  • the expression vector comprises a nucleic acid sequence coding the FV or derivative thereof polypeptide, or a functional fragment thereof as described herein.
  • Administration of FV or derivative thereof-encoding expression vectors to a patient results in the expression of FV or derivative thereof polypeptide which serves to enhance coagulation.
  • the FV or derivative thereof encoding nucleic acid sequence may encode FV or derivative thereof polypeptide as described herein whose expression modulates hemostasis.
  • Expression vectors comprising FV or derivative thereof nucleic acid sequences may be administered alone, or in combination with other molecules useful for modulating hemostasis. According to the present invention, the expression vectors or combination of therapeutic agents may be administered to the patient alone or in a pharmaceutically acceptable or biologically compatible composition.
  • the expression vector comprising nucleic acid sequences encoding FV or derivative thereof is a viral vector.
  • Viral vectors which may be used in the present invention include, but are not limited to, adenoviral vectors (with or without tissue specific promoters/enhancers), adeno-associated virus (AAV) vectors of multiple serotypes (e.g., AAV-2, AAV-5, AAV-7, and AAV-8) and hybrid AAV vectors, lentivirus vectors and pseudo-typed lentivirus vectors [e.g., Ebola virus, vesicular stomatitis virus (VSV), and feline immunodeficiency virus (FIV)], herpes simplex virus vectors, vaccinia virus vectors, and retroviral vectors.
  • Adenoviral vectors of utility in the methods of the present invention preferably include at least the essential parts of adenoviral vector DNA. As described herein, expression of FV or derivative thereof polypeptide following administration of such an adenoviral vector serves to modulate hemostasis.
  • adenoviral vectors have found broad utility for a variety of gene therapy applications. Their utility for such applications is due largely to the high efficiency of in vivo gene transfer achieved in a variety of organ contexts.
  • Adenoviral particles may be used to advantage as vehicles for adequate gene delivery.
  • Such virions possess a number of desirable features for these applications, including: structural features related to being a double stranded DNA nonenveloped virus and biological features such as a tropism for the human respiratory system and gastrointestinal tract.
  • adenoviruses are known to infect a wide variety of cell types in vivo and in vitro by receptor-mediated endocytosis. Attesting to the overall safety of adenoviral vectors, infection with adenovirus leads to a minimal disease state in humans which comprises mild flu-like symptoms.
  • adenoviral genomes Due to their large size (-36 kilobases), adenoviral genomes are well suited for use as gene therapy vehicles because they can accommodate the insertion of foreign DNA following the removal of adenoviral genes essential for replication and nonessential regions. Such substitutions render the viral vector impaired with regard to replicative functions and infectivity.
  • adenoviruses have been used as vectors for gene therapy and for expression of heterologous genes.
  • adenovirus vectors utilized for gene therapy, see Berkner, 1988, Biotechniques 6:616-629 and Trapnell, 1993, Advanced Drug Delivery Reviews 12: 185-199. It is desirable to introduce a vector that can provide, for example, multiple copies of a desired gene and hence greater amounts of the product of that gene. Improved adenoviral vectors and methods for producing these vectors have been described in detail in a number of references, patents, and patent applications, including: Mitani and Kubo (2002, Curr Gene Ther. 2(2): 135-44); Olmsted- Davis et al. (2002, Hum Gene Ther. 13(11): 1337-47); Reynolds et al.
  • an expression construct may further comprise regulatory elements which serve to drive expression in a particular cell or tissue type.
  • regulatory elements are known to those of skill in the art and discussed in depth in Sambrook et al. (1989) and Ausubel et al. (1992).
  • tissue specific regulatory elements are known to those of skill in the art and discussed in depth in Sambrook et al. (1989) and Ausubel et al. (1992).
  • tissue specific regulatory elements are known to those of skill in the art and discussed in depth in Sambrook et al. (1989) and Ausubel et al. (1992).
  • the incorporation of tissue specific regulatory elements in the expression constructs of the present invention provides for at least partial tissue tropism for the expression of FV or derivative thereof or functional fragments thereof.
  • an El deleted type 5 adenoviral vector comprising nucleic acid sequences encoding FV or derivative thereof under the control of a cytomegalovirus (CMV) promoter may be used to advantage in the methods of the present invention.
  • CMV
  • Adenoviral vectors for recombinant gene expression have been produced in the human embryonic kidney cell line 293 (Graham et al., 1977, J, Gen. Virol. 36:59- 72). This cell line is permissive for growth of adenovirus 2 (Ad2) and adenovirus 5 mutants defective in El functions because it comprises the left end of the adenovirus 5 genome and, therefore, expresses El proteins. El genes integrated into the cellular genome of 293 cells are expressed at levels which facilitate the use of these cells as an expression system in which to amplify viral vectors from which these genes have been deleted.
  • 293 cells have been used extensively for the isolation and propagation of El mutants, for helper-independent cloning, and for expression of adenovirus vectors.
  • Expression systems such as the 293 cell line, therefore, provide essential viral functions in trans and thereby enable propagation of viral vectors in which exogenous nucleic acid sequences have been substituted for El genes. See Young et al. in The Adenoviruses, Ginsberg, ed., Plenum Press, New York and London (1984), pp. 125-172.
  • Other expression systems well suited to the propagation of adenoviral vectors are known to those of skill in the art (e.g., HeLa cells) and have been reviewed elsewhere.
  • Also included in the present invention is a method for modulating hemostasis comprising providing cells of an individual with a nucleic acid delivery vehicle encoding FV or derivative thereof polypeptide and allowing the cells to grow under conditions wherein the FV or derivative thereof polypeptide is expressed. From the foregoing discussion, it can be seen that variant FV or derivative thereof polypeptide or nucleic acids encoding the same may be used in the treatment of disorders associated with aberrant blood coagulation.
  • the expression vectors of the present invention may be incorporated into pharmaceutical compositions that may be delivered to a subject, so as to allow production of a biologically active protein (e.g., f FV or derivative thereof polypeptide or functional fragment or derivative thereof).
  • a biologically active protein e.g., f FV or derivative thereof polypeptide or functional fragment or derivative thereof.
  • pharmaceutical compositions comprising sufficient genetic material to enable a recipient to produce a therapeutically effective amount of FV or derivative thereof polypeptide can influence hemostasis in the subject.
  • an effective amount of the variant Factor V polypeptide may be directly infused into a patient in need thereof.
  • compositions may be administered alone or in combination with at least one other agent, such as a stabilizing compound, which may be administered in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water.
  • a stabilizing compound which may be administered in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water.
  • the compositions may be administered to a patient alone, or in combination with other agents which influence hemostasis.
  • the pharmaceutical compositions also contain a pharmaceutically acceptable excipient.
  • excipients include any pharmaceutical agent that does not itself induce an immune response harmful to the individual receiving the composition, and which may be administered without undue toxicity.
  • Pharmaceutically acceptable excipients include, but are not limited to, liquids such as water, saline, glycerol, sugars and ethanol.
  • Pharmaceutically acceptable salts can also be included therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles.
  • mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like
  • organic acids such as acetates, propionates, malonates, benzoates, and the like
  • auxiliary substances such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles.
  • compositions suitable for parenteral administration may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiologically buffered saline.
  • Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
  • suspensions of the active compounds may be prepared as appropriate oily injection suspensions.
  • Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes.
  • the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
  • penetrants appropriate to the particular barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
  • the pharmaceutical compositions of the present invention may be manufactured in any manner known in the art (e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing processes).
  • the pharmaceutical composition may be provided as a salt and can be formed with many acids, including but not limited to, hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents than are the corresponding, free base forms.
  • the preferred preparation may be a lyophilized powder which may contain any or all of the following: 1-50 ⁇ M histidine, l%-2% sucrose, and 2-7% mannitol, at a pH range of 4.5 to 5.5, which is combined with buffer prior to use.
  • compositions suitable for use in the invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended therapeutic purpose. Determining a therapeutically effective dose is well within the capability of a skilled medical practitioner using the techniques provided in the present invention. Therapeutic doses will depend on, among other factors, the age and general condition of the subject, the severity of the aberrant blood coagulation phenotype, and the strength of the control sequences regulating the expression levels of FV or derivative thereof polypeptide. Thus, a therapeutically effective amount in humans will fall in a relatively broad range that may be determined by a medical practitioner based on the response of an individual patient to vector-based form of or protein infused FV or derivative thereof treatment.
  • variant activated form of FV polypeptide alone or in combination with other agents may be directly infused into a patient in an appropriate biological carrier as described hereinabove.
  • Expression vectors of the present invention comprising nucleic acid sequences encoding the activated form of FV or derivative thereof, or functional fragments thereof, may be administered to a patient by a variety of means (see below) to achieve and maintain a prophylactically and/or therapeutically effective level of the activated form of FV or derivative thereof polypeptide.
  • One of skill in the art could readily determine specific protocols for using the activated form of FV or derivative thereof encoding expression vectors of the present invention for the therapeutic treatment of a particular patient.
  • adenoviral vectors Protocols for the generation of adenoviral vectors and administration to patients have been described in U.S. Patent Nos. 5,998,205; 6,228,646; 6,093,699; 6,100,242; and International Patent Application Nos. WO 94/17810 and WO 94/23744., which are incorporated herein by reference in their entirety.
  • the activated form of FV or derivative thereof encoding adenoviral vectors of the present invention may be administered to a patient by any means known.
  • Direct delivery of the pharmaceutical compositions in vivo may generally be accomplished via injection using a conventional syringe, although other delivery methods such as convection-enhanced delivery are envisioned (See e.g., U.S. Pat. No. 5,720,720).
  • compositions may be delivered subcutaneously, epidermally, intradermally, intrathecally, intraorbitally, intramucosally, intraperitoneally, intravenously, intraarterially, orally, intrahepatically or intramuscularly.
  • Other modes of administration include oral and pulmonary administration, suppositories, and transdermal applications.
  • a clinician specializing in the treatment of patients with blood coagulation disorders may determine the optimal route for administration of the adenoviral vectors comprising the activated form of FV or derivative thereof nucleic acid sequences based on a number of criteria, including, but not limited to: the condition of the patient and the purpose of the treatment (e.g., enhanced blood coagulation).
  • compositions of the invention may be used to induce coagulation topically for wounds and traumatic injury.
  • the Factor V of the invention will be formulated for topical application and used on the battlefield or under circumstances in the field where bleeding must stopped or significantly stanched.
  • the present invention also encompasses AAV vectors comprising a nucleic acid sequence encoding the activated form of FV or derivative thereof polypeptide.
  • lentivirus or pseudo-typed lentivirus vectors comprising a nucleic acid sequence encoding an activated form of FV or derivative thereof polypeptide
  • naked plasmid or expression vectors comprising a nucleic acid sequence encoding an activated form of FV or derivative thereof polypeptide.
  • the inhibitors benzamidine and 4-amidinophenylmethanesulfonyl fluoride hydrochloride were from Sigma (St. Louis, MO) and dansylarginine-N-(3 -ethyl- l,5-pentanediyl)amide (DAPA) was from Haematologic Technologies (Essex Junction, VT).
  • the peptidyl substrate H-D- phenylalanyl-L-pipecolyl-L-arginyl-p-nitroanilide (S2238) was from Diapharma (West Chester, OH).
  • tissue culture reagents were from Invitrogen (Carlsbad, CA), except insulin-transferrin- sodium selenite (ITS; Roche Applied Science, Indianapolis, IN).
  • ITS insulin-transferrin- sodium selenite
  • PCPS Small unilamellar phospholipids vesicles
  • 75% (w/w) hen egg L- ⁇ -phosphatidylcholine and 25% (w/w) porcine brain L- ⁇ -phosphatidylserine were prepared and characterized as described (22).
  • Human prothrombin was isolated from plasma as described previously (23). Prethrombin-1 and prethrombin-2 were purified by established procedures (18), and thrombin was obtained from Haematologic Technologies. A constitutively active partial B-domainless form of FV (hFV-810), rFVa, rFX and rFXa were prepared, purified and characterized as described (3, 24, 25). The purified enzymatic subunit of pseutarin C (pt-FXa) was a generous gift from QRxPharma (Sydney, Australia). Recombinant hirudin was obtained from EMD-Chemicals (San Diego, CA). Recombinant tick anticoagulant protein and human APC were obtained from Dr.
  • Sedimentation velocity was measured at 20°C in 20 mM Hepes, 150 mM NaCl, 5 mM CaCl 2 , pH 7.4 at 25,000 rpm with an AN60Ti rotor. Protein concentrations were: pt- rFV, 0.60 mg/mL (3.6 ⁇ M) and pt-FXa, 0.18 mg/mL (3.9 ⁇ M). Sedimentation coefficients and molecular weights were determined by g(s*) analysis (28). Extinction coefficients were determined by differential refractometry by the procedure described by Babul and Stellwagen (29).
  • rFV variants The P. textilis FV cDNA derived from the venom gland (8) was modified with flanking Xmal restriction sites and then subcloned into the pED expression plasmid (3). The cDNA was further modified by exchanging sequences encoding the signal sequence of P. textilis FV with that of human FV using the technique of splicing by overlap extension (30). The final construct, pED-FV-ptet, encoded for the human FV signal peptide (residues -28 to -1) followed by the mature venom-derived pt-FV protein (residues 1-1430). Sequence analysis indicated that the P.
  • Plasmids encoding pt-FV or pt-FV-QQ were introduced into baby hamster kidney (BHK) cells and high producing stable clones were established as described (3). Cells were expanded into triple flasks and cultured in DMEM/F12 media supplemented with ITS and 2.5 mM CaCl 2 .
  • Conditioned media was collected for 4-6 days, centrifuged, and stored at -20 0 C in the presence of 10 ⁇ M APMSF and 1 mM Benzamidine.
  • media was thawed at 37 0 C and loaded onto a 30 mL Q-Sepharose FF column (Amersham Biosciences, Piscataway, NJ) equilibrated in 20 mM Hepes, 0.15 M NaCl, 5 mM CaCl 2 , pH 7.4. The column was washed with the same buffer and eluted with a 0.15-1 M NaCl gradient. Fractions were collected and analyzed by SDS-PAGE and by a FV- specific clotting assay as described (3).
  • Fractions containing pt-rFV were pooled and dialyzed versus 20 mM MES, 2 mM CaCl 2 , pH 6.0. and then loaded on a Poros HS/20 column (10 x 100 mm; Applied Biosystems, Foster City, CA) equilibrated with the same buffer. The column was washed with 20 mM MES, 2 mM CaCl 2 , pH 6.0 and then eluted with a 0-0.6 M NaCl gradient. FV containing fractions were pooled and dialyzed versus 20 mM Hepes, 0.15 M NaCl, 2 mM CaCl 2 , pH 7.4, and the protein was stored at -80 0 C. The final yield was ⁇ 4 mg of pt-rFV/liter of conditioned media.
  • Protein characterization Protein purity was assessed by SDS-PAGE using pre-cast 4- 12% gradient gels (Invitrogen) under non-reducing and reducing conditions (50 mM dithiothreitol) employing the MOPS buffer system followed by staining with Coomassie Brilliant Blue R-250. N-terminal sequence analysis was performed in the laboratory of Dr. Alex Kurosky and Steve Smith at the University of Texas Medical Branch at Galveston or Dr. Jan Pohl, Center for Disease Control (Atlanta, Ga). Chemical ⁇ - carboxyglutamic acid (GIa) analysis on pt-FXa was carried out in the laboratory as described (24).
  • GIa ⁇ - carboxyglutamic acid
  • Kinetics of protein substrate cleavage Steady state initial velocities of macromolecular substrate cleavage were determined discontinuously at 25 0 C as described (25).
  • the kinetic parameters of prothrombinase or FXa-FVa- catalyzed prothrombin or prethrombin-1 activation were determined in assay buffer by measuring the initial rate of thrombin formation at increasing concentrations of macromolecular substrate.
  • Assay mixtures contained PCPS (50 ⁇ M), DAPA (3 ⁇ M), the various pt-rFV(a) cofactor species (20 nM), and various concentrations of prothrombin (0-5.0 ⁇ M) or prethrombin-1 (0-15 ⁇ M).
  • the reaction was initiated with pt-FXa (0.1 nM).
  • prethrombin-2 was used as protein substrate, the following reaction conditions were used: PCPS (50 ⁇ M), DAPA (3 ⁇ M), and prethrombin-2 (1.4 ⁇ M) were incubated with the various FV(a) cofactors (0-40 nM), and the reaction was initiated with pt-FXa (5 nM).
  • PCPS was omitted from the reaction mixture (see figure legends).
  • pt-rFVa pt-rFV (600 nM) was incubated with 2 nM thrombin at 37 0 C for 15 min followed by the addition of 2.4 nM hirudin.
  • Assay mixtures contained, 1.4 ⁇ M prothrombin, 50 ⁇ M PCPS, 3 ⁇ M DAPA, 1 nM FV(a), and 1 nM pt-FXa. With rhFVa or hFV-810, human rFXa was used instead. Under these conditions, the initial rate of the reaction was proportional to the concentration of the cofactor.
  • the present invention describes the biological properties of snake venom derived FV from P. textilis. These unique properties, listed below, could also be engineered into a human FV derivative recapitulating the procoagulant properties. These properties are: a) pt-FV exists in an active, cofactor state and is as such the first example of a naturally occurring active FV variant; b) pt-FV has a unique conformation as it bypasses the normal requirement for a membrane surface to achieve high affinity binding to its serine protease factor Xa (FXa); c) pt-FV is functionally resistant to inactivation by activated protein C (APC), due to a unique disulfide bond in pt-FV that connects its heavy and light chains. Engineering a human FV derivative comprising one or more of these characteristics will enhance its procoagulant properties, thereby providing a useful therapeutic tool for treatment of bleeding disorders. In the present study, a series of functional measurements with recombinant venom-
  • FV pt-rFV
  • pt-FXa highly purified venom-derived FXa
  • the findings shed new light on multiple aspects of FV structure/function relationships, facilitate bioengineering FV for therapeutic purposes, and also highlight the remarkable ability of nature to enhance the existing properties of a protein for selective advantage.
  • pt-rFV-QQ was not cleaved by thrombin
  • pt-rFV was processed to pt-rFVa, yielding the characteristic heavy and light chains.
  • N-terminal sequence analysis revealed correct processing at the predicted thrombin cleavage sites (Fig. 1) (7, 8).
  • a large fraction (>60%) of pt-rFVa migrated as a single band under non-reducing conditions (Fig. 1 , panel B), indicating that the heavy and light chains are held together by a unique disulfide bond. This likely accounts for migration of pt-rFV and pt-rFV-QQ as doublets under non-reducing conditions.
  • Sequence alignment suggest that pt-FV likely has two free Cys residues in the heavy chain (A2 domain, 539 and 641) and one free Cys in the light chain (A3 domain, 1032) (7).
  • FV purified from the crude venom of P. textilis or O. scutellatns has low activity in the presence of bovine FXa (14, 15).
  • the initial attempts to characterize pt-rFV were consistent with these observations.
  • the low activity appeared to be principally due to the finding that pt-rFV or pt-rFVa bound with a reduced affinity to membrane-bound human FXa (K ⁇ i >400 nM) using established fluorescent binding measurements (data not shown) (3).
  • pt- FXa was used in all subsequent experiments.
  • a central view of blood coagulation is that membrane binding of coagulation proteins to cellular surfaces (via phosphatidylserine exposure) is critical for normal hemostasis (2).
  • An unusual observation made with oscutarin and pseutarin C is that venom-derived FXa and FV co-purify as a complex and exhibit some level of activity in the absence of anionic membranes; however, the activity of oscutarin is reported to be substantially increased (>400-fold) in the presence of membranes (12-15). To explore this further, the activity of solution phase pt-FXa-pt-rFV using prothrombin was assessed.
  • a difficulty in interpreting these data is that the substrate, prothrombin, binds membranes contributing to enhancing rates of thrombin generation. Furthermore, depending on the solution phase dissociation constant for pt-FXa-pt-rFV, pt-FXa may not have been saturated with pt-rFV, reducing the actual enzyme complex concentration, leading to a decreased rate of prothrombin activation. To address the first point, the rate of prethrombin-1 activation in the presence and absence of membranes was measured. Prethrombin-1 is a derivative of prothrombin lacking the membrane binding GIa and kringle-1 domains (18).
  • Pt-rFV is functionally resistant to APC
  • APC is an important anticoagulant in the hemostatic system and functions by inactivating both FVa and FVIIIa via limited proteolysis (21).
  • FVa cleavage at Arg 306 , Arg 506 , and Arg 679 results in A2 domain dissociation, loss of FXa binding and ultimately cofactor function (1).
  • Sequence analysis of pt-FV indicates that these sites are not well conserved suggesting it could be resistant to APC (7).
  • Studies with pseutarin C support this contention (7); however, FXa can protect FVa from inactivation by APC complicating data interpretation (1).
  • Rao VS, Kini RM (2002) Pseutarin C, a prothrombin activator from Pseudonaja textilis venom: its structural and functional similarity to mammalian coagulation factor Xa-Va complex. Thromb Haemost 88:611-619. 16.
  • Rao VS, Swarup S, Kini RM (2004) The catalytic subunit of pseutarin C, a group C prothrombin activator from the venom of Pseudonaja textilis, is structrually similar to mammalian blood coagulation factor Xa. Thromb Haemost 92:509-521.
  • Boskovic DS Giles AR, Nesheim MD (1990) Studies of the role of factor Va in the factor Xa- catalyzed activation of prothrombin, fragment 1 2-prethrombin-2, and dansyl-1-glutamyl- glycyl-1-arginine-meizothrombin in the absence of phospholipid. J Biol Chem 265 : 10497-

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Zoology (AREA)
  • Toxicology (AREA)
  • Hematology (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Diabetes (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Public Health (AREA)
  • Engineering & Computer Science (AREA)
  • Veterinary Medicine (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)
  • Medicinal Preparation (AREA)

Abstract

La présente invention concerne des compositions et des procédés pour le traitement de troubles de la coagulation utilisant des variants de facteur V. Les troubles préférés comprennent l'hémophilie A et B.
EP09828344.3A 2008-11-21 2009-11-23 Facteur v de serpent et procédés d'utilisation en tant que procoagulant Active EP2365822B1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US11691308P 2008-11-21 2008-11-21
PCT/US2009/065515 WO2010060035A1 (fr) 2008-11-21 2009-11-23 Facteur v de serpent et procédés d'utilisation en tant que procoagulant

Publications (3)

Publication Number Publication Date
EP2365822A1 true EP2365822A1 (fr) 2011-09-21
EP2365822A4 EP2365822A4 (fr) 2013-03-27
EP2365822B1 EP2365822B1 (fr) 2015-07-01

Family

ID=42198533

Family Applications (1)

Application Number Title Priority Date Filing Date
EP09828344.3A Active EP2365822B1 (fr) 2008-11-21 2009-11-23 Facteur v de serpent et procédés d'utilisation en tant que procoagulant

Country Status (6)

Country Link
US (1) US8557762B2 (fr)
EP (1) EP2365822B1 (fr)
JP (1) JP2012509670A (fr)
AU (1) AU2009316380B2 (fr)
CA (1) CA2744174C (fr)
WO (1) WO2010060035A1 (fr)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2019344801A1 (en) * 2018-09-17 2021-05-20 Board Of Regents, The University Of Texas System Compositions and methods for treating bone injury

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007101106A2 (fr) * 2006-02-23 2007-09-07 The Children's Hospital Of Philadelphia Compositions et méthodes pour moduler une hémostase au moyen de différentes formes de facteur v activé
WO2009079690A1 (fr) * 2007-12-20 2009-07-02 The University Of Queensland Compositions modulant l'hémostase et utilisations associées

Family Cites Families (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6093699A (en) 1987-07-09 2000-07-25 The University Of Manitoba Method for gene therapy involving suppression of an immune response
WO1994017810A1 (fr) 1993-02-12 1994-08-18 The Wistar Institute Of Anatomy And Biology Vaccin recombine contre le cytomegalovirus
WO1994023744A1 (fr) 1993-04-16 1994-10-27 The Wistar Institute Of Anatomy And Biology Vaccin contenant un cytomegalovirus de recombinaison
AU7676894A (en) 1993-08-27 1995-03-21 Government Of The United States Of America, As Represented By The Secretary Of The Department Of Health And Human Services, The Convection-enhanced drug delivery
AT401270B (de) 1994-09-26 1996-07-25 Immuno Ag Verfahren zur quantifizierung von genomischer dna
US5998205A (en) 1994-11-28 1999-12-07 Genetic Therapy, Inc. Vectors for tissue-specific replication
CN100569297C (zh) 1995-02-28 2009-12-16 加利福尼亚大学董事会 基因转移介导的血管形成疗法
US20030143759A1 (en) 1995-10-20 2003-07-31 Bjorn Dahlback Assays for determining anticoagulant cofactor activity
WO1997032481A1 (fr) 1996-03-07 1997-09-12 The Regents Of The University Of California Adenovirus sans assistant et totalement defectif pour therapie genetique

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007101106A2 (fr) * 2006-02-23 2007-09-07 The Children's Hospital Of Philadelphia Compositions et méthodes pour moduler une hémostase au moyen de différentes formes de facteur v activé
WO2009079690A1 (fr) * 2007-12-20 2009-07-02 The University Of Queensland Compositions modulant l'hémostase et utilisations associées

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
M. H. A. BOS ET AL: "Venom factor V from the common brown snake escapes hemostatic regulation through procoagulant adaptations", BLOOD, vol. 114, no. 3, 16 July 2009 (2009-07-16) , pages 686-692, XP055053424, ISSN: 0006-4971, DOI: 10.1182/blood-2009-02-202663 *
RAO V S ET AL: "The Nonenzymatic Subunit of pseutarin C, a Prothrombin Activator from Eastern Brown Snake (Pseudonoja textiles) Venom, Shows Structural Similarity to Mammalian Coagulation Factor V", BLOOD, AMERICAN SOCIETY OF HEMATOLOGY, US, vol. 102, no. 4, 15 August 2003 (2003-08-15), pages 1347-1354, XP008138406, ISSN: 0006-4971 *
See also references of WO2010060035A1 *

Also Published As

Publication number Publication date
US20110268790A1 (en) 2011-11-03
AU2009316380A1 (en) 2010-05-27
EP2365822B1 (fr) 2015-07-01
US8557762B2 (en) 2013-10-15
JP2012509670A (ja) 2012-04-26
EP2365822A4 (fr) 2013-03-27
CA2744174C (fr) 2017-06-06
CA2744174A1 (fr) 2010-05-27
AU2009316380B2 (en) 2016-02-04
WO2010060035A1 (fr) 2010-05-27

Similar Documents

Publication Publication Date Title
KR101574042B1 (ko) 지혈을 조절하기 위한 조성물 및 방법
US10106786B2 (en) Compositions and methods for modulating hemostasis
US10676731B2 (en) Compositions and methods for modulating factor IX function
US8557762B2 (en) Snake factor V and methods of use thereof as a procoagulant
JP2019525764A (ja) 第ｉｘ因子の機能を調節するための組成物及び方法
WO2023212539A1 (fr) Compositions et procédés de modulation de la fonction du facteur viii

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20110616

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO SE SI SK SM TR

DAX Request for extension of the european patent (deleted)
REG Reference to a national code

Ref country code: DE

Ref legal event code: R079

Ref document number: 602009031997

Country of ref document: DE

Free format text: PREVIOUS MAIN CLASS: A61K0038260000

Ipc: A61K0038360000

A4 Supplementary search report drawn up and despatched

Effective date: 20130227

RIC1 Information provided on ipc code assigned before grant

Ipc: C07K 14/745 20060101ALI20130221BHEP

Ipc: A61K 38/36 20060101AFI20130221BHEP

Ipc: C07K 14/46 20060101ALI20130221BHEP

17Q First examination report despatched

Effective date: 20140327

GRAP Despatch of communication of intention to grant a patent

Free format text: ORIGINAL CODE: EPIDOSNIGR1

INTG Intention to grant announced

Effective date: 20141217

GRAS Grant fee paid

Free format text: ORIGINAL CODE: EPIDOSNIGR3

GRAA (expected) grant

Free format text: ORIGINAL CODE: 0009210

RAP1 Party data changed (applicant data changed or rights of an application transferred)

Owner name: THE CHILDREN'S HOSPITAL OF PHILADELPHIA

AK Designated contracting states

Kind code of ref document: B1

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO SE SI SK SM TR

REG Reference to a national code

Ref country code: GB

Ref legal event code: FG4D

REG Reference to a national code

Ref country code: AT

Ref legal event code: REF

Ref document number: 733534

Country of ref document: AT

Kind code of ref document: T

Effective date: 20150715

Ref country code: CH

Ref legal event code: EP

REG Reference to a national code

Ref country code: IE

Ref legal event code: FG4D

REG Reference to a national code

Ref country code: DE

Ref legal event code: R096

Ref document number: 602009031997

Country of ref document: DE

REG Reference to a national code

Ref country code: CH

Ref legal event code: NV

Representative=s name: ISLER AND PEDRAZZINI AG, CH

REG Reference to a national code

Ref country code: FR

Ref legal event code: PLFP

Year of fee payment: 7

REG Reference to a national code

Ref country code: AT

Ref legal event code: MK05

Ref document number: 733534

Country of ref document: AT

Kind code of ref document: T

Effective date: 20150701

REG Reference to a national code

Ref country code: NL

Ref legal event code: MP

Effective date: 20150701

REG Reference to a national code

Ref country code: LT

Ref legal event code: MG4D

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: GR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20151002

Ref country code: LT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20150701

Ref country code: NO

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20151001

Ref country code: LV

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20150701

Ref country code: FI

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20150701

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: SE

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20150701

Ref country code: PL

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20150701

Ref country code: IS

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20151101

Ref country code: HR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20150701

Ref country code: ES

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20150701

Ref country code: PT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20151102

Ref country code: AT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20150701

REG Reference to a national code

Ref country code: DE

Ref legal event code: R097

Ref document number: 602009031997

Country of ref document: DE

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: EE

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20150701

Ref country code: SK

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20150701

Ref country code: DK

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20150701

Ref country code: IT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20150701

Ref country code: CZ

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20150701

PLBE No opposition filed within time limit

Free format text: ORIGINAL CODE: 0009261

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: NO OPPOSITION FILED WITHIN TIME LIMIT

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: RO

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20150701

26N No opposition filed

Effective date: 20160404

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: LU

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20151123

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: SI

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20150701

REG Reference to a national code

Ref country code: FR

Ref legal event code: PLFP

Year of fee payment: 8

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: BE

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20150701

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: BG

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20150701

Ref country code: HU

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT; INVALID AB INITIO

Effective date: 20091123

Ref country code: SM

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20150701

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: CY

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20150701

Ref country code: NL

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20150701

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: TR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20150701

Ref country code: MT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20150701

REG Reference to a national code

Ref country code: FR

Ref legal event code: PLFP

Year of fee payment: 9

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: MK

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20150701

REG Reference to a national code

Ref country code: FR

Ref legal event code: PLFP

Year of fee payment: 10

P01 Opt-out of the competence of the unified patent court (upc) registered

Effective date: 20230526

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: IE

Payment date: 20230912

Year of fee payment: 15

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: FR

Payment date: 20230929

Year of fee payment: 15

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: GB

Payment date: 20231006

Year of fee payment: 15

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: MC

Payment date: 20231027

Year of fee payment: 15

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: DE

Payment date: 20230926

Year of fee payment: 15

Ref country code: CH

Payment date: 20231201

Year of fee payment: 15